Field of the Invention
The invention generally relates to the field of immunology.
Background Art
The natural immune system strikes a complex balance between highly aggressive, protective immune responses to foreign pathogens and the need to maintain tolerance to normal tissues. In recent years there has been increasing recognition that interactions among many different cell types contribute to maintaining this balance. Such interactions can, for example, result in polarized responses with either production of pro-inflammatory cytokines by TH1 type T cells or production of interleukin-4 (IL4) by TH2 type T cells that suppress TH1 activity. In a number of different animal models, T cell polarization to TH1 has been shown to favor protective immunity to tumors or infectious pathogens whereas T cell polarization to TH2 can be a critical factor in preventing development of cell-mediated autoimmune disease. The conditions that determine whether immune stimulation will result in aggressive cell-mediated immunity or in down regulation of such responses are highly localized in the sense that each tissue is comprised of a distinctive set of antigen presenting cells (APC) and lymphocyte lineages that interact to favor different immune responses. For example, under optimal conditions, the dendritic cells (DC) localized in a normal tissue may represent predominantly a lineage and stage of maturation that favors tolerogenic interactions and serves as a barrier to cell-mediated autoimmunity whereas a tumor or site of infection will attract mature myeloid dendritic cells that stimulate potent cell-mediated immune responses.
CD1d-restricted NKT cells are a unique class of non-conventional T cells that appear to play an important role in defining the outcome of immune stimulation in the local environment. They share with the larger class of NKT cells the expression of markers of both the T cell and natural killer (NK) cell lineages although several studies suggest that expression of NK markers such as CD161 and NKG2d by NKT cells is a function of both stage of maturation and state of activation (Chen et al., J Immunol 1997; 158: 5112-9). As such, NKT cells are considered as part of innate immunity like NK cells and in humans their frequency in normal individuals can be as high as 2.0% of total T lymphocytes (Gumperz et al., 2002. J Exp Med 195:625; Lee et al., 2002. J Exp Med 195:637).
CD1d-restricted NKT cells are distinguished from other NKT cells by their specificity for lipid and glycolipid antigens presented by the monomorphic MHC class Ib molecule, CD1d (Kawano et al., Science 278 (1997), pp. 1626-16292). CD1d is a non-MHC encoded molecule that associates with β2-microglobulin and is structurally related to classical MHC class I molecules. In contrast to MHC class I and class II molecules that sample peptides from the cytosol and endocytic compartments and transport them to the cell surface where they can be recognized by T cells, CD1d has a hydrophobic antigen-binding pocket that is specialized for binding the hydrocarbon chains of lipid tails or hydrophobic peptides (Zeng et al., Science 277 (1997), pp. 339-345). CD1d is known to bind a marine sponge derived α-glycosylated sphingolipid, α-galactosylceramide (α-GalCer), and related molecules such as sphingolipids with α-linked galactose or glucose but not mannose (Kawano et al., Science 278 (1997), pp. 1626-1629; and Zeng et al., Science 277 (1997), pp. 339-345). As discussed below, the ability to activate many CD1d-restriced NKT cells by stimulation with α-GalCer or related molecules bound to CD1d of antigen presenting cells has greatly facilitated functional analysis of this non-conventional T cell subset. In the absence of inflammation, CD1d-restricted NKT cells have been shown to localize preferentially in certain tissues like thymus, liver and bone marrow (Wilson et al., 2002. Trends Mol Med 8:225) and antitumor activity of NKT cells has been mainly investigated in mouse liver metastasis.
NKT cells have an unusual ability of secreting both TH1 and TH2 cytolines and potent cytotoxic as well as regulatory functions have been documented in inflammation, autoimmunity and tumor immunity (Bendelac et al., 1995 Science 268:863; Chen and Paul. 1997. J Immunol 159:2240; and Exley et al., 1997. J Exp Med 186:109). Distinct functional subsets of NKT cells have been characterized as regards their cytoline profiles, expression of several NK receptors, tissue segregation and CD1d dependance (Gumperz et al., 2002. J Exp Med 195:625; Lee et al., 2002. J Exp Med 195:637; Eberl et al., 1999. J Immunol 162:6410; and MacDonald, 2002, Curr Opin Immunol 14:250).
Among the CD1d-restricted NKT cells is a subset that expresses a highly conserved αβT cell receptor (TCR). In man this invariant TCR is comprised of Vα24Jα15 in association with Vβ11 whereas in mice the receptor comprises the highly homologous Vα14Jα18 and Vβ8.2. Other CD1d-restricted NKT cells express more variable TCR Both TCR invariant and TCR variant classes of CD1d-restricted T cells can be detected by binding of CD1d-tetramers loaded with α-GalCer (Benlagha et al., J Exp Med 191 (2000), pp. 1895-1903; Matsuda et al., J Exp Med 192 (2000), pp. 741-754; and Karadimitris et al., Proc Natl Acad Sci USA 98 (2001), pp. 3294-3298). CD1d-restricted NKT cells, as defined in this application (CD1d-NKT), include cells that express either invariant or variant TCR and that bind or are activated by CD1d loaded with either α-GalCer or with related sphingolipids that have α-linked galactose or glucose including molecules such as OCH, which differs from α-GalCer by having a shortened long-chain sphingosine base (C5 vs. C14) and acyl chain (C24 vs. C26) (Miyamoto et al., Nature 2001 413:531-4). A common feature of CD1d-NKT cells is that, in contrast to conventional T cells, these lymphocytes often have a surface phenotype (CD44hiCD62L-CD69+) that is characteristic of recently activated or memory T cells. This striking phenotype has been explained by an in vivo autoreactivity of these T cells to still unknown autologous ligands presented by CD1d (Kronenberg and Gapin. 2002 Nat Rev Immunol 2:557).
CD1d-NKT have been shown to have direct cytotoxic activity against targets that express CD1d. It is likely, however, that the effect of CD1d-NKT on immune responses is amplified through recruitment of other lymphocytes either by direct interaction or, perhaps even more importantly, by indirect recruitment through interaction with DC. CD1d-NKT have the unique ability to secrete large quantities of IL-4 and IFN-γ early in an immune response. Secretion of IFN-γ induces activation of DC which produce interleukin-12 (IL-12). IL-12 stimulates further IFN-γ secretion by NKT cells and also leads to activation of NK cells which secrete more IFN-γ.
Since CD1d-NKT are able to rapidly secrete large amounts of both IL-4 and IFN-γ, the polarization of immune responses will depend on whether the effect of pro-inflammatory IFN-γ or anti-inflammatory IL-4 cytokines predominate. This has been reported to be, in part, a function of the relative frequency of different subsets of CD1d-NKT. These subsets include (i) an invariant CD1d-NKT population that is negative for both CD4 and CD8 and that gives rise to predominantly a TH1 type response including secretion of pro-inflammatory IFN-γ and TNF-α and (ii) a separate population of CD1d-NKT that is CD4+ and that gives rise to both a TH1 type and TH2 type response including secretion of the anti-inflammatory Th2-type cytokines IL-4, IL-5, IL-10 and IL-13 (Lee et al., J Exp Med 2002; 195: 637-41; and Gumperz et al., J Exp Med 2002; 195: 625-36). Local factors that influence activation of CD1d-NKT subsets include the cytokine environment and, importantly, the DC that are recruited to that environment.
The availability of a defined antigen, αC-GalCer, that can be employed to specifically activate the CD1d-NKT cell subset has made it possible to examine the role of these non-conventional T cells in a variety of immune responses. Administration of the α-GalCer lipid antigen has a dramatic effect on a number of different microbial infections, including protective effects in murine malaria, fungal and hepatitis B virus infections (Kakimi et al, J Exp Med 192 (2000), pp. 921-930; Gonzalez-Aseguinolaza et al., Proc Natl Acad Sci USA 97 (2000), pp. 8461-8466; and Kawakami et al., Infect Immun 69 (2001), pp. 213-220). Dramatic effects of administration of α-GalCer have also been observed in animal models of tumor immunity. Stimulation with α-GalCer or with cytokines like IL-12 suppresses lung and liver metastases in an NKT dependent manner as shown by loss of protection in mice that do not develop CD1d-NKT because they are deficient in CD1d or in the NKT TCRα chain that is dominantly expressed in CD1d-NKT (Smyth et al., 2002. Blood 99:1259; Hayakawa et al., Eur J Immunol 31:1720; Takeda et al., Int Immunol 12:909). One study (Cui et al., Science 278 (1997), pp. 1623-1626) demonstrated that the antitumor response to melanomas observed in tumor-bearing mice treated with IL-12 is solely due to NKT cells. In contrast to normal mice with CD1d-NKT cells, IL-12 treated tumor-bearing mice deficient in the Jα15 gene (which are deficient in NKT cells, since most mouse CD1d-NKT cells express Jα15 positive invariant TCR) could not control B16 melanoma growth and metastases.
A number of indirect mechanisms contribute to the protective effect of CD1d-NKT cells. Activation of NKT cells by administration of α-GalCer in vivo results in concomitant activation of NK cells (Eberl and MacDonald, Eur. J. Immunol. 30 (2000), pp. 985-992; and Carnaud et al., J. Immunol. 163 (1999), pp. 4647-4650). In mice deficient in NKT cells, α-GalCer is unable to induce cytotoxic activity by NK cells. NKT cells also enhance the induction of classical MHC class I restricted cytotoxic T cells (Nishimura et al., Int Immunol 2000; 12: 987-94; and Stober et al., J Immunol 2003; 170:2540-8).
The participation of NKT cells at the earliest stages of the protective immune response to many pathogens (infections (Kakimi et al, J Exp Med 192 (2000), pp. 921-930; Gonzalez-Aseguinolaza et al., Proc Natl Acad Sci USA 97 (2000), pp. 8461-8466; Kawakami et al., Infect Immun 69 (2001), pp. 213-220; and Bendelac and Medzhitov, J Exp Med 2002; 195: F19-23) and tumors (Kobayashi et al., Oncol Res 1995; 7: 529-34; and Smyth et al., Curr Opin Immunol 2002; 14:165-71) is a reflection of their direct cytotoxic activity as well as their important contribution to general mobilization of an aggressive cell-mediated immune response. Extensive evidence suggests, however, that NKT cells also function to suppress autoimmunity (Hong et al., Nature Med 2001; 7: 1052-6; Beaudoin et al., Immunity 2002; 17: 725-36; Wilson et al., Trends Mol Med 2002; 8: 225-31; Shi et al., Proc Natl Acad Sci USA 2001; 98: 6777-82; Naumov et al., Proc Natl Acad Sci USA 2001; 98: 13838-43; Sharif et al., Nature Med 2001; 7: 1057-62; Wang et al., J Exp Med 2001; 194: 313-20; Jahng et al., J Exp Med 2001; 194: 1789-99; Singh et al., J Exp Med 2001; 194:1801-11), maintain immune privilege (Sonoda et al., J Exp Med 1999; 190: 1215-26; Hong and Van Kaer, J Exp Med 1999; 190: 1197-1200), and support engraftment of transplanted tissues (Seino et al., Proc Natl Acad Sci USA 2001; 98: 2577-81; Zeng et al., J Exp Med 1999; 189: 1073-81). These include experiments in which administration of α-GalCer has been shown to protect against autoimmune diabetes in non-obese diabetic (NOD) mice (Hong et al., Nature Med 2001; 7: 1052-6; Wilson et al., Trends Mol Med 2002; 8: 225-31; Sharif et al., Nature Med 2001; 7: 1057-62) and against experimental autoimmune encephalomyelitis (EAE), a murine model of demyelinating disease (Jahng et al., J Exp Med 2001; 194: 1789-99; and Singh et al., J Exp Med 2001; 194: 1801-11). Conversely, deletion of NKT cells results in exacerbation or potentiation of disease in these models (Shi et al., Proc Natl Acad Sci USA 2001; 98: 6777-82). Parallel studies of the frequency of CD1d-NKT cells in human cancer and certain autoimmune diseases have demonstrated that a deficiency in such T cells is associated with progressive disease (Tahir et al. J. Immunol. 167:4046-50,2001; Sharif et al. J. Mol. Med. 80:290-300,2002; Sumidha et al. J. Exp. Med. 182:1163-68,1995; Mes et al. J. Immunol. 164:4375-81,2000; van der Vliet et al. Clin. Immunol. 100:144-148,2001). In contrast, some autoimmune diseases, including myasthenia gravis (Reinhardt et al. Neurology 52:1485-87,1999), psoriasis (Bonish, J. Immunol. 165:4076-85,2000), ulcerative colitis (Saubermann et al. Gastroenterology 119:119-128, 2000), and primary biliary cirrhosis (Kita et al. Gastroenterology 123:1031-43,2002) may have an etiology that reflects excessive IL-4 and IFN-γ production by CD1d-NKT cells. In the case of psoriasis this appears to be related to overexpression of CD1d in keratinocytes of chronic, active psoriatic plaques.
The divergent pro-inflammatory and anti-inflammatory effects of CD1d-NKT cells in different circumstances have been attributed to different functional subsets of CD1d-NKT and dendritic cells and to differences in the representation of these subsets in the local tissue environment. An interesting example is a study demonstrating that transfer of myeloid DC derived from pancreatic lymph nodes, but not from inguinal lymph nodes, of mice treated systemically with α-GalCer completely protects NOD female mice from diabetes [30]. As a result of this potential for divergent effects, systemic activation of CD1d-NKT, for example by administration of α-GalCer, may have an undesirable outcome in the treatment of specific disease. There is a need, therefore, for a means of targeted activation of CD1d-NKT in a specific local environment and/or by targeted interaction with a defined subset of dendritic cells.